Protease stability

In Fig. 3 the sequences of 12 modified as well as two natural peptides which serve as control sequences are given [54]. The diastereomers of all Tfm-substituted peptides were separately studied regarding their protease stability. The results of this hydrolysis study are summarized in Fig. 4. 

Tetrahedral borate or boronate complexes have been shown to be involved in enzyme inhibition. Serine proteases were proposed to be inhibited by boric acid [6], and simple borates have been patented as protease stabilizers in liquid detergent formulations [7, 8]. 

Meng, H. and Kumar, K. (2007) Antimicrobial activity and protease stability of peptides containing fluorinated amino acids. Journal of the American Chemical Society, 129(50), 15615-15622. 

The patent literature contains numerous examples of enzyme stabilization schemes based on the use of various chemical additives. Common themes are to either lower the amount of water in the formulation while retaining solubility of other components, or to add specific enzyme stabilizer/inhibitors. Some examples are highlighted in Table 4, and many others can be found in the review by Crutzen and Douglass. Few published articles have addressed the mechanisms of enzyme stabilization by additives. One study examines protease stabilization by carboxylic acid salts and another discusses the use of borate in conjunction with propylene glycol to inhibit protease activity. ... 

Crossin, M.C. Protease Stabilization by carboxylic-acid salts - relative efficiencies and mechanisms. Journal of the American Oil Chemists Society 1989, 66 (7), 1010-1014. 

Lalonde, J. Witte, E.J. Oconnell, M.L. Holliday, L. Protease stabilization by highly concentrated anionic surfactant mixtures. Journal of the American Oil Chemists Society 1995, 72 (1), 53-59. 

Russell, G.L. Britton, L.N. Use of certain alcohol ethoxylates to maintain protease stability in the presence of anionic surfactants. Journal of Surfactants and Detergents 2002, 5 (1), 5-10. 

Citric acid itself has also been patented as an ingredient in protease stabilization systems [91]. 

FIG. 8.21 Effect of product pH on protease stability in a HDLD containing alcohol ethoxylate and alcohol ethoxy sulfates. (Reproduced from Kravetz, L. and Guin, K.F., J. Am. Oil Chem. Soc., 62, 943, 1985. With permission.)... 

Letton and Yunker [104] and Kaminsky and Christy [105] describe protease stabilization systems comprised of a combination of a calcium salt and a salt of a carboxylic acid, preferably a formate. These ingredients are moderately effective in enzyme stabilization and are relatively inexpensive. Care has to be taken, however, when adding divalent ions such as calcium to HDLDs to prevent the possibility of precipitation. 

Although the nature of the peptoid backbone is quite similar to the nature of the peptide backbone, these new unnatural oligomers display some peculiar characteristics (i) the peptoid backbone is achiral (ii) peptoids are devoid of amide protons, which decrease their polarity and should increase their oral bioavailability (iii) they lack the NH-donor bond and, therefore, they do not have the possibility to form intra- or intermolecular H-bonding through the backbone (iv) they can be more flexible and adopt altered conformations and (v) they contain only tertiary amide bonds, which display a higher protease stability [7],... 

Sainz, C.B., Younce, F.L., Rasco, B., and Clark, S. 2009. Protease stability in bovine milk under combined thermal-high hydrostatic pressure treatment. Innovative Food Science and Emerging Technologies 10 314-320. 

While linear peptides have been used extensively in the very early stages of lead finding (cf. Chapter 1.4) they found only limited application as therapeutic agents due to their limited protease stability (and thus low plasma stability and bioavailability) and low penetration through cell membranes. To circumvent some of these inherent bioavailability problems associated with linear peptides several approaches have been published to modify and stabilise linear peptides by construction of peptide mimetics. " Figure 2.3.1 schematically summarises these different approaches. 

The major difference between peptides and peptoids lies in the fact that the side chain substituents R -R in peptoids are attached to the amide nitrogens, thus avoiding the chiral a-centres of a peptide. There are no NH-bonds present in peptoids which has major implications on solubility, cell penetration properties and on the overall conformations which differ significantly from those of peptides. In addition, compared to peptides, peptoids show an increased protease stability due to the tertiary amide bond. 

X-ray crystallographic studies of serine protease complexes with transition-state analogs have shown how chymotrypsin stabilizes the tetrahedral oxyanion transition states (structures (c) and (g) in Figure 16.24) of the protease reaction. The amide nitrogens of Ser and Gly form an oxyanion hole in which the substrate carbonyl oxygen is hydrogen-bonded to the amide N-H groups. 

In the first publication describing the preparative use of an enzymatic reaction in ionic liquids, Erbeldinger et al. reported the use of the protease thermolysin for the synthesis of the dipeptide Z-aspartame (Entry 6) [34]. The reaction rates were comparable to those found in conventional organic solvents such as ethyl acetate. Additionally, the enzyme stability was increased in the ionic liquid. The ionic liquid was recycled several times after the removal of non-converted substrates by extraction with water and product precipitation. Recycling of the enzyme has not been reported. It should be noted, however, that according to the log P concept described in the previous section, ethyl acetate - with a value of 0.68 - may interfere with the pro-... 

Roth, A. F., and Ward, W. W. (1983). Conformational stability after protease treatment in Aequorea GFP. Photochem. Photobiol. 37S S71. 

The family of serine proteases has been subjected to intensive studies of site-directed mutagenesis. These experiments provide unique information about the contributions of individual amino acids to kcat and KM. Some of the clearest conclusions have emerged from studies in subtilisin (Ref. 9), where the oxyanion intermediate is stabilized by t>e main-chain hydrogen bond of Ser 221 and an hydrogen bond from Asn 155 (Ref. 2). Replacement of Asn 155 (e.g., the Asn 155— Ala 155 described in Fig. 7.9) allows for a quantitative assessment of the effect of the protein dipoles on Ag. ... 

The prehminary data suggests that aPNAs are stable towards degradation by the proteases present in human serum. Further studies are required to determine the stability of aPNAs in vivo. 

Further, Wasserman and coworkers developed a direct acylation of stabilized phosphonium ylides by carboxylic acids in presence of the EDCI/DMAP (way c). This last method allows the introduction of a-aminoacid structures into the resulting P-oxo phosphorus ylides [19-25],opening the way to the total synthesis of depsipeptide elastase inhibitors [22,24] or cyclic peptidic protease inhibitor EurystatinA [20]. 

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